Ferredoxin (Fd) is the major iron-containing protein in photosynthetic organisms and is central to reductive metabolism in the chloroplast. The Chlamydomonas reinhardtii genome encodes six plant type [Fe 2 S 2 ] ferredoxins, products of PETF, FDX2-FDX6. We performed the functional analysis of these ferredoxins by localizing Fd, Fdx2, Fdx3, and Fdx6 to the chloroplast by using isoform-specific antibodies and monitoring the pattern of gene expression by iron and copper nutrition, nitrogen source, and hydrogen peroxide stress. In addition, we also measured the midpoint redox potentials of Fd and Fdx2 and determined the kinetic parameters of their reactions with several ferredoxin-interacting proteins, namely nitrite reductase, Fd:NADP ؉ oxidoreductase, and Fd:thioredoxin reductase. We found that each of the FDX genes is differently regulated in response to changes in nutrient supply. Moreover, we show that Fdx2 (E m ‫؍‬ ؊321 mV), whose expression is regulated by nitrate, is a more efficient electron donor to nitrite reductase relative to Fd. Overall, the results suggest that each ferredoxin isoform has substrate specificity and that the presence of multiple ferredoxin isoforms allows for the allocation of reducing power to specific metabolic pathways in the chloroplast under various growth conditions.Ferredoxins are small (ϳ11,000-kDa), soluble, iron-sulfur cluster-containing proteins with strongly negative redox potentials (Ϫ350 to Ϫ450 mV) that function as electron donors at reductive steps in various metabolic pathways (1-3). In photosynthetic organisms, the well studied ferredoxin (Fd 4

Although resistin has been thought to be an important link between obesity and diabetes, recent results do not support this hypothesis. We speculated that resistin may be involved in in£ammatory processes and be induced by in£amma-tory stimuli. In this study, we tested whether lipopolysaccharide (LPS) induced resistin expression in rats. The results show that resistin mRNA levels in white adipose tissue and white blood cells were increased by LPS treatment. LPS also increased resistin mRNA levels in 3T3-L1 adipocytes and human peripheral blood monocytes. The results suggest that resistin is involved in insulin resistance and probably in other in£ammatory responses.

To elucidate the importance of aflatoxin in the etiology of hepatocellular carcinoma (HCC), a community-based cohort study combined with molecular dosimetry of aflatoxin exposure was performed in the Penghu Islets where the HCC mortality rate is highest in Taiwan. A total of 6,487 residents aged 30 to 65 years were recruited in the two-stage screening survey and underwent regular follow-up examination. Among 33 newly diagnosed HCC cases, 31 (94%) were chronic hepatitis B surface antigen (HBsAg) carriers and 3 (9%) were positive for antibodies against hepatitis C virus (HCV). Among 20 HCC patients and 86 matched healthy controls whose serum samples were tested for aflatoxin B1 (AFB1)-albumin adducts by competitive enzyme-linked immunosorbant assay (ELISA), 13 (65%) HCC patients and 32 (37%) matched controls were seropositive, showing a statistically significant multivariate-adjusted odds ratio of 5.5 with a 95% confidence interval of 1.2 to 24.5. The results imply the elevated risk of HCC among Penghu residents may be attributable to their heavy exposure to aflatoxins and high HBsAg carrier rate.

Two unlinked genes FER1 and FER2 encoding ferritin subunits were identified in the Chlamydomonas genome. An improved FER2 gene model, built on the basis of manual sequencing and incorporation of unplaced reads, indicated 49% identity between the ferritin subunits. Both FER1 and FER2 transcripts are increased in abundance as iron nutrition is decreased but the pattern for each gene is distinct. Using subunitspecific antibodies, we monitored expression at the protein level. In response to low iron, ferritin1 subunits and the ferritin1 complex are increased in parallel to the increase in FER1 mRNA. Nevertheless, the iron content of the ferritin1 complex is decreased. This suggests that increased expression results in increased capacity for iron binding in the chloroplast of iron-limited cells, which supports a role for ferritin1 as an iron buffer. On the other hand, ferritin2 abundance is decreased in iron-deprived cells, indicative of the operation of iron-nutrition-responsive regulation at the translational or post-translational level for FER2. Both ferritin subunits are plastid localized but ferritin1 is quantitatively recovered in soluble extracts of cells while ferritin2 is found in the particulate fraction. Partial purification of the ferritin1 complex indicates that the two ferritins are associated in distinct complexes and do not coassemble. The ratio of ferritin1 to ferritin2 is 70:1 in iron-replete cells, suggestive of a more dominant role of ferritin1 in iron homeostasis. The Volvox genome contains orthologs of each FER gene, indicating that the duplication of FER genes and potential diversification of function occurred prior to the divergence of species in the Volvocales.A LTHOUGH iron is abundant on earth, its bioavailability is limited in the aerobic world because of the insolubility of ferric salts, and iron can be a limiting nutrient for most forms of life. A third of the agricultural land and the same fraction of the ocean are considered iron deficient (reviewed by Boyd et al. 2007). Therefore, iron nutrition is a key component of global productivity. Organisms have evolved multiple and varied pathways for assimilating iron in its various chemical forms and at a range of concentrations in the nutrient environment (Staiger 2002; reviewed in Curie and Briat 2003;Hentze et al. 2004). Even though iron can be toxic to cells as a consequence of its propensity for participating in redox chemistry, cells do not generally excrete iron because it is a limiting nutrient (Liochev and Fridovich 1999). Instead, cells tend to store intracellular iron in a less reactive and hence nontoxic form. Because of the importance of iron for life, these pathways of uptake and storage are subject to layers of homeostatic regulation.We have developed Chlamydomonas as a model organism for understanding trace metal nutrition in plants, especially in the context of chloroplast function and photosynthesis (Merchant et al. 2006). As a microorganism, Chlamydomonas lends itself to nutritional studies because of the ease wit...

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Hypercholesterolemic human LDL contains oxidized subfractions that have atherogenic properties. Paradoxically, atherosclerosis incidence is low in patients with primary biliary cirrhosis (PBC), a disease characterized by marked increases in plasma LDL, including the LDL subfraction lipoprotein-X (Lp-X). To investigate the mechanisms underlying this paradox, we first examined the propensity to oxidation of unfractionated LDL isolated from PBC patients. After prolonged incubation with copper, PBC-LDL failed to increase the oxidation index or electrophoretic mobility noted in control LDL. An admixture of PBC-LDL or Lp-X with control LDL prevented oxidation of the latter in a dose-dependent manner. PBC-LDL was also noncompetitive against copper-oxidized LDL (oxLDL) for binding with a murine monoclonal anti-oxLDL antibody in a competitive ELISA. OxLDL exerts its proapoptotic and antiangiogenic effects in part by inhibiting fibroblast growth factor 2 (FGF2) expression. Preincubation of oxLDL with PBC-LDL, but not control LDL, attenuated the inhibitory effects of oxLDL on FGF2 expression in cultured bovine aortic endothelial cells (ECs). The antioxidant and prosurvival properties of PBC-LDL diminished after the patients underwent orthotopic liver transplantation. These results suggest that Lp-X reduces LDL atherogenicity by preventing LDL oxidation to protect EC integrity in the presence of hypercholesterolemia. They also suggest that altering LDL composition may be as important as reducing LDL concentration in preventing or treating atherosclerosis. -Chang, P-Y., S-C. Lu, T-C. Su, S-F. Chou, W-H. Huang, J. D. Morrisett, C-H. Chen, C-S. Liau, and Y-T. Lee. Lipoprotein-X reduces LDL atherogenicity in primary biliary cirrhosis by preventing LDL oxidation.

Hepatitis C virus (HCV) is a positive-stranded RNA virus classified in the Hepacivirus genus in the family Flaviviridae. The 9.6-kb viral RNA genome encodes a precursor polyprotein that is processed to generate at least 10 viral proteins, including structural proteins (core, E1, E2, and p7) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (1). The NS5B RNA polymerase, together with other nonstructural viral proteins (NS3, NS4A, NS4B, and NS5A) and host factors, constitutes the active replication complexes (RC) for viral RNA replication. These proteins are directly or indirectly associated with the endoplasmic reticulum (ER)-derived structure called the "membranous web," where replication occurs (2, 3). However, the exact host factors and detailed interactions within the RC remain to be determined.HCV infection usually causes chronic hepatitis and frequently leads to cirrhosis and hepatocellular carcinoma (HCC). Besides, HCV-induced end-stage liver disease is an important indication for liver transplantation in most of the Western countries (4, 5). At present, no effective vaccine to prevent HCV infection is available. The current treatment strategies for HCV infection are valid only for individuals with a particular single nucleotide polymorphism (SNP) in the interleukin-28B gene or for infections caused by certain viral genotypes (6). Accordingly, the prevalence of hepatic steatosis in HCV-infected patients is much higher than that in the general population or in hepatitis B virus (HBV)-infected patients (7). Hepatic steatosis has also been reported to be associated with an increased rate of HCC in chronic hepatitis C patients (8).Lipid metabolic pathways are essential for the entry, secretion, and replication of HCV. For example, apolipoprotein E (apoE) is essential for the production of HCVcc (HCV produced in cell culture) and for viral entry (9, 10). Downregulation of apoA-I decreases levels of HCV replication and viral particle production in cell culture (11). HCV coopts the secretory pathway of very low density lipoprotein (VLDL) for its own secretion (12, 13). Moreover, HCV replication is regulated through induction of lipogenic gene expression in HCV replicon cells (14) or geranylgeranylation of host proteins required for HCV RNA replication (15,16). Fatty acid synthesis is also required for HCV RNA replication (14). Inhibition of fatty acid synthase (FASN) by cerulenin (17) or C75 (18) reduces the replication of subgenomic HCV replicons as well as JFH-1-based HCVcc virion production. Although the underlying mechanisms are not yet completely understood, these studies imply that FASN is essential for HCV replication.In this study, HCV NS5B was used as the bait to screen for NS5B-interacting proteins that are present in Huh7 hepatoma cell lysates. After mass spectrophotometric analysis, FASN was found to interact with NS5B, and this interaction was further confirmed in vitro and in vivo. Our data indicate that FASN interacts with NS5B to enhance NS5B RNA-dependent RNA polymerase (RdRp) act...

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